Cadophora species from marine glaciers in the Qinghai-Tibet Plateau: an example of unsuspected hidden biodiversity

Large numbers of marine glaciers in the Qinghai-Tibet Plateau are especially sensitive to changes of climate and surface conditions. They have suffered fast accumulation and melting and retreated quickly in recent years. In 2017, we surveyed the cold-adapted fungi in these unique habitats and obtained 1208 fungal strains. Based on preliminary analysis of ITS sequences, 41 isolates belonging to the genus Cadophora were detected. As one of the most frequently encountered genera, the Cadophora isolates were studied in detail. Two phylogenetic trees were constructed: one was based on the partial large subunit nrDNA (LSU) to infer taxonomic placement of our isolates and the other was based on multi-locus sequences of LSU, ITS, TUB and TEF-1α to investigate more exact phylogenetic relationships between Cadophora and allied genera. Combined with morphological characteristics, nine Cadophora species were determined, including seven new to science. Among the new species, only C. inflata produces holoblastic conidia and all the others express phialidic conidiogenesis. All isolates have optimum growth temperature at 20 °C or 25 °C. With more species involved, the currently circumscribed genus became obviously paraphyletic. All members are clustered into two main clades: one clade mainly includes most of the Cadophora species which have phialidic conidiogenesis and we refer to as ‘Cadophora s. str.’; the remaining Cadophora species have multiform conidiogenesis and are clustered in the second clade, with members of other genera in Ploettnerulaceae interspersed among the subclades. The results show a high diversity of Cadophora from marine glaciers in the Qinghai-Tibet Plateau and most of them are novel species.


Introduction
The genus Cadophora was first established in 1927, with C. fastigiata as the type species, to accommodate dematiaceous hyphomycetes producing solitary phialides with distinct hyaline collarettes (Lagerberg et al. 1927). Due to subtle differences in morphology, Conant (1937) transferred eight Cadophora species to the genus Phialophora.
Later, Gams (2000) proposed that Phialophora species related to discomycete sexual morphs of Mollisia and related genera belonging to Helotiales should be accommodated in Cadophora. This proposal was supported by subsequent rDNA sequence analysis of LSU (Harrington and McNew 2003).
Currently, the genus is included in the family Ploettnerulaceae of Helotiales (Johnston et al. 2019;Ekanayaka et al. 2019) and comprises some species with multiform morphological characters deviated from the original morphological generic concept. For example, C. antarctica and C. fascicularis produce chains of ramoconidia and conidia on holoblastic conidiogenous cells (Crous

IMA Fungus
*Correspondence: wangqm@hbu.edu.cn; wangmm@hbu.edu.cn Page 2 of 29 Zhang et al. IMA Fungus (2022) 13:15 et al. 2017;Maciá-Vicente et al. 2020); while C. obovata has putatively monoblastic conidiogenous cells that may represent a retrogression of enteroblastic phialidic conidiogenesis and C. fallopiae is only observed as a cladophialophora-like synasexual morph in culture (Maciá-Vicente et al. 2020;Crous et al. 2020). Besides, C. lacrimiformis only found by its sexual morph, is also included in this asexually typified genus (Ekanayaka et al. 2019). Recent studies based on molecular data have shown that Cadophora is apparently paraphyletic and species with distinct morphological variations may share ancestors with other related genera (Maciá-Vicente et al. 2020). Species of Cadophora normally possess multiple trophic modes. They are commonly considered as plant pathogens, root associates or wood and soil colonizers with cosmopolitan distribution. A global survey on the dominant soil fungal communities of different biomes has shown that Cadophora is one of the most ubiquitous soil fungal taxa with significantly higher number of genes related to stress-tolerance and resource uptake (Egidi et al. 2019). In some cold Arctic and Antarctic sites, Cadophora species have been frequently isolated from soils, marine sediments and organisms, fresh water lakes, especially the historic wood huts and some mummified or submerged drift wood (Blanchette et al. 2004(Blanchette et al. , 2010(Blanchette et al. , 2016Jurgens et al. 2009;Gonçalves et al. 2012;Furbino et al. 2014;Zhang et al. 2017;Nagano et al. 2017;Duran et al. 2019). They are hypothesized to be key organisms capable of initiating nutrient cycles and energy flows from dead organic materials in high latitudes (Blanchette et al. 2016). Meanwhile, the saprotrophic species, mainly C. malorum, C. luteo-olivacea, and C. fastigiata which were frequently isolated from polar regions are also detected as pathogens or endophytes from different living plants worldwide (Di Marco et al. 2004;Gramaje et al. 2011;Navarrete et al. 2011;Travadon et al. 2015). Enzyme tests of some Cadophora members have shown that C. luteo-olivacea and C. malorum are capable of degrading a range of carbon sources and releasing soluble phosphorus so that their trophic modes could vary depending on their nutrient needs from different substrata (Day and Currah 2011;Walsh et al. 2018).
The Qinghai-Tibet Plateau, lying across the center of Asia and having an average elevation of 4000 m, possesses large numbers of glacial groups that constitute the center of Asian Highland Glaciers. Based on hydrothermal conditions and physical properties, glaciers in China can be divided into continental glaciers and marine glaciers. Continental glaciers, which are also known as cold glaciers, develop in the continental climate areas where precipitation amount is limited; marine glaciers, which are also known as temperate glaciers, generally form in marine climate areas with abundant precipitation (Shi et al. 1964(Shi et al. , 2000. Controlled by the marine monsoonal climate, nearly 9000 marine glaciers, which cover a total area of 13,200 square kilometers and account for 22.2% of the total glacier area in China, form at southeast margin of the Qinghai-Tibet Plateau. Under the background of global warming, glaciers all over the world are retreating significantly. In the next 100 years, marine glaciers in the Qinghai-Tibet Plateau, with the features of fast accumulation and melting and being more sensitive to the change of climate, will retreat more quickly (Yao et al. 2004;Chen et al. 2005). It is necessary and urgent to investigate fungal diversity and resources in this unique area.
Our first investigation (2009-2011) on cold-adapted fungi in the permafrost and alpine glaciers of Qinghai-Tibet Plateau indicates that the diversity of cold-adapted fungi from marine glaciers is especially high and many of them may represent unknown species (Wang et al. 2015). Another survey was conducted in 2017, focusing on the diversity of cold-adapted fungi from marine glaciers. Based on preliminary analyses of the generated ITS sequences, 41 strains representing nine Cadopora species including seven new species are described and phylogenetic relationships intra and among Cadophora and related genera are discussed in this study.

Sample collection
Soil, ice and water samples were collected from four marine glaciers and two nearby snow-capped mountains in 2017 (Table 1). Sampling sites were selected at different elevations of the following marine glaciers and snow-capped mountains: Hailuogou Glacier, Yanzigou Glacier and Dagu Glacier in Sichuan Province, Yulong Snow Mountain, Baima Snow Mountain and Mingyong Glacier in Yunnan Province (Figs. 1,2). For all samplings, clean hand tools were surface sterilized with 70% ethanol before use. After the removal of the top 5-10 cm of surface sediment, c. 500 g soil or ice sample was collected from the underlying layer and placed in a fresh Zip-lock plastic bag and sterilized plastic bottles. Melt water samples were directly collected and placed in sterilized centrifuge tubes or Zip-lock plastic bags. All the samples were maintained at 4 °C until arrival at the laboratory.

Isolation
Strains were isolated from soil and water samples as soon as they were taken to the lab. Soil samples were isolated with traditional pour plate method: A 10 g quantity of each soil sample was suspended in steriledistilled water in a flask, the volume was then increased to 100 mL before the suspension was shaken to disperse soil particles and then serially diluted to 10 -2 , 10 -3 and 10 -4 ; 100 mL of each water sample was filtrated by nitrocellulose filter membrane with pore size of 0.45 μm, the membrane with trapped fungi was put in a sterile 50 mL centrifuge tube which contained 10 mL distilled water and the tube was vigorously agitated to suspend the trapped mycelium and spores. About 0.1 mL of each final diluent or concentrate was placed on the surface of two 90 mm diam Petri plates containing 1/4 strength Potato Dextrose Agar (1/4 PDA; 9 g of Potato Dextrose Agar [BD Difco] and 15 g of Agar per L of demineralized water) supplemented with chloramphenicol (0.1 mg/mL) and streptomycin (0.1 mg/mL). The plates were sealed and incubated at 15 °C and 25 °C (one plate per temperature) and were examined for fungal growth at 1 wk intervals for 4 wk. Colonies that appeared on the plates were transferred to two new plates and then incubated at 15 °C and 25 °C. All fungal strains were stored at 4 °C for further studies.

Morphological studies
41 isolates representing all of the Cadophora species isolated were studied in more detail. To enhance sporulation, strains were inoculated on potato dextrose agar (PDA; BD Difco), malt extract agar (MEA, BD Difco) and oatmeal agar (OA; BD Difco). Pine needle medium, H 2 O 2 treatment and slide culture technique (Xu et al. 2009;Su et al. 2012) were also used to induce sporulation. For phenotypic determination, the strains were transferred to PDA, MEA and OA plates with three replicates and incubated in the dark at 25 °C. Optimal growth temperature (OGT) and maximum growth temperature (MGT) were also tested by culturing each isolate in triplicate on PDA at temperatures ranging from 5 to 35 °C at 5 °C increments. Colony diameters were measured in two perpendicular directions after 2 wk at different temperatures, and the mean diameter was obtained from three replicate plates cultivated at the same temperatures. Colony colors were determined using taxonomic description color charts (Rayner 1970). Microscopic preparations were made by mounting aerial hyphae in water or using the slide cultures directly. Hyphae, conidiophores, and conidia were observed, photographed, and measured with 1000 × magnification by using a Nikon 80i microscope with differential interference contrast (DIC) optics. Specimens were deposited in the Mycological Herbarium of Hebei University (HBU), while living cultures including ex-types were deposited in the China General Microbiological Culture Collection Center (CGMCC).

DNA extraction, PCR amplification, sequencing and phylogenetic analyses
Genomic DNA was extracted from the fungal mycelia following the protocol described by Wang and Zhuang (2004). The partial large subunit nrDNA (LSU), the Page 4 of 29 Zhang et al. IMA Fungus (2022) 13:15 internal transcribed spacer region of the nuclear ribosomal RNA gene (ITS), the partial translation elongation factor 1-α gene (TEF-1α) and the β-tubulin (β-TUB) gene were amplified and sequenced with the primer pairs of LROR/LR5 (Vilgalys and Hester 1990), ITS1/ITS4 (White et al. 1990), EF1-688F/EF1-1251R (Alves et al. 2008)   Page 6 of 29 Zhang et al. IMA Fungus (2022) 13:15 were sequenced with the primers mentioned above by BGI Tech Solutions Co., Ltd. (Shenzhen, China). Nucleotide sequences were initially checked and edited using Chromas software ver. 2.6.6 (http:// www. techn elysi um. com. au/ chrom as. html) and EdiSeq (Lasergene, DNASTAR) and then were compared to accessions in the GenBank database via BLASTn searching to find the most likely taxonomic designation. To reveal the family placements of the species described in this study, a LSU tree was constructed. To investigate more exact phylogenetic relationships and taxonomic distinctions of novel species, a multi-locus analysis was performed based on ITS, LSU, TUB and TEF1-α genes. Sequence data of the four genes especially those of ex-type strains, were downloaded from GenBank and added to the sequences generated in this study. The datasets were aligned automatically using MAFFT v. 7.471 (Katoh and Standley 2013) and further manual alignment was carried out with MEGA v. 7 (Kumar et al. 2016) and alignments were deposited in TreeBASE (www. treeb ase. org, submission no. S29383).
Phylogenetic analyses were conducted using Bayesian Inference (BI), Maximum Likelihood (ML) and Maximum Parsimony (MP) methods. For BI analyses, the best fit model of evolution for each partition was estimated by MEGA v. 7. Posterior probabilities were determined by Markov Chain Monte Carlo sampling (MCMC) in MrBayes v. 3.2.7a (Ronquist and Huelsenbeck 2003) using the estimated models of evolution. For the LSU/multilocus trees, six simultaneous Markov chains were run for 4,000,000/8,000,000 generations and trees were sampled every 100th generation (resulting in 40,000/80,000 total trees). The first 10,000/20,000 trees represented the burn-in phase of the analyses were discarded and the remaining 30,000/60,000 trees were used for posterior probabilities (PP) calculation in the majority rule consensus trees. The ML analyses were performed by raxmlGUI 2.0.0-beta (Edler et al. 2019) using the GTRGAMMA model with the rapid bootstrapping and search for best scoring ML tree algorithm, including 1000 bootstrap replicates. The MP analyses were conducted using PAUP v. 4.0b10 (Swofford 2002) and an unweighted parsimony (UP) analysis was performed. Trees were inferred using the heuristic search option with TBR branch swapping and 1000 random sequence additions. Branches of zero length were collapsed and all equally most parsimonious trees were saved. Descriptive tree statistics such as tree length (TL), consistency index (CI), retention index (RI), rescaled consistency index (RC) and homoplasy index (HI), were calculated for trees generated. Clade stability was assessed using bootstrap analysis with 1000 replicates, each with 10 replicates of random stepwise addition of taxa.

Results
1208 fungal strains isolated from 120 samples of four glaciers and two snow-capped mountains were preliminarily identified based on BLAST comparison of ITS sequences against the GenBank database. As one of the most commonly encountered fungal groups, 41 isolates belonging to Cadophora were studied in detail.

Phylogenetic analyses
Sequences of referential species, especially those of extype strains, were retrieved from GenBank and added to the sequences generated in this study ( Table 2). The alignments of partial sequences of LSU (For LSU phylogenetic analysis), ITS, LSU (For muti-locus phylogenetic analysis), TUB and TEF1-α have 855, 452, 834, 582 and 694 characters, respectively.
According to the LSU phylogenetic tree, representative Cadophora strains of this study (marked with bold font) and the known Cadophora species are interspersed with species of other genera in Ploettnerulaceae and form a well-supported clade (BP/BP/PP = 90/98/100, ML/MP bootstrap and BI posterior probability support values, respectively) that distinctly separate from other family members in the Helotiales (Fig. 3).
A multi-gene phylogenetic tree is also employed to investigate further phylogenetic relationships intra and among Cadophora and allied genera (Fig. 4). All the representative species cluster into two main clades with high ML/MP bootstrap or BI posterior probability support values (85/100/100, 97/100/100 respectively). In the first main clade (Clade 1), 38 isolates of this study form six distinct subclades: isolates of YZ1026 and YZ1034 cluster in a lineage including the ex-type sequences of C. novi-eboraci with strong branch support; although strain MY902 and the known species of Hymenula cerealis form a well-supported subclade, they are obviously distinguished morphologically. The placement of H. cerealis should also be confirmed by protein coding genes which are currently unavailable; the other four subclades group seperately from previously described species. Combined with morphological characteristics, we propose five Cadophora species new to science: Cadophora caespitosa, C. daguensis, C. indistincta, C. magna and C. qinghai-tibetana. Clade 1 also includes most of the phialidic Cadophora species (including the type species of the genus) and three species (Hymenula cerealis, Mollisia cinerella and Phialophora dancoi) currently placed in other genera. The second main clade (Clade 2) contains the remaining Cadophora species and most of the other Ploettnerulaceae members. Three isolates of this study are included in this clade: strain YL412 clusters with C. malorum in a well supported lineage; strain MY759 and       (Fig. 5).
Colonies on MEA flat, primrose to pale citrine, white at the margin, reverse same colours. Colonies on OA with a yellow margin, surface blackbrown, aerial mycelium sparse, reverse same colours. Colonies on PDA with a distinct and smooth margin, flat, grey to red, white at the edge, reverse dark-red. Page 17 of 29 Zhang et al. IMA Fungus (2022) 13:15 Notes: Cadophora indistincta is phylogenetically related to C. qinghai-tibetana (Fig. 4), but they are especially different in colours of colony on PDA and the length of collarettes (Figs. 8,13). Cadophora indistincta produces red coloured colony on PDA and this is also a distinct character different from other Cadophora species except C. ferruginea, but the colour of the colony produced by C. ferruginea is rust red and darker than that of C. indistincta. Hyphal cells often strongly inflated, up to 6-10 μm wide, form chains or microsclerotia-like bodies. Conidiophores very short or highly reduced. Conidiogenous cells holoblastic. Conidia hyaline, attached to mycelium, located laterally or terminally, smooth-walled, globular or spathulate, solitary, 2.9-7.1 × 3.0-4.4 μm (mean = 3.9 ± 0.8 × 3.7 ± 0.4 μm, n = 30), L/W ratio = 1.1. Culture characteristics -Colonies on MEA reaching 28 mm diam, after 14 d at 25 °C in the dark, on OA and PDA reaching 47 mm and 37 mm diam,  Zhang et al. IMA Fungus (2022) 13:15 respectively. OGT 25 °C and MGT over 35 °C (Fig. 5). Colonies on MEA, with an entire margin, flat, white, lacking aerial mycelium, reverse same colours. Colonies on OA with a smooth margin, flat, black in the center, olivaceous to white from middle to edge, reverse same colours. Colonies on PDA with a smooth margin, felty, grey, pale yellow at the margin, reverse grey-brown with a pale buff to white margin. Notes: Cadophora inflata is characterized by chains or microsclerotia-like inflated cells that are similar to Leptodophora gamsii and L. echinata which were first described as C. gamsii and C. echinata (Maciá-Vicente et al. 2020). The original authors interpreted these structures as holoblastic conidia but they may just as well described as inflated hyphal segments with dormancy functions. Our newly described species failed to produce conidia on MEA, OA, and PDA media. We also tried other methods such as treating the cultures with H 2 O 2 or culturing the isolates on pine needle medium before a slide culture technique was used. Cadopohra inflata produces globose or ellipsoidal conidia attached directly to the hyphae with very short conidiophores that resemble those of Leptodophora orchidicola, which has been transferred from Cadophora to Leptodophora (Koukol & Description: Mycelium hyaline to dark brown, septate, branched, smooth-walled, 1-3 μm, hyphal cells often strongly inflated, variable in shape. Conidiophores brown, smooth-walled, often reduced to conidiogenous cells. Conidiogenous cells phialidic, mostly single, arranged terminally or laterally on the hyphae, cylindrical to navicular, apex wedge, base truncate, smooth-walled, straight or slightly curved, 12.7-20.3 × 2.8-3.8 μm, collarettes funnelshaped, 1.9-3.0 μm long, opening 2.8-2.9 μm wide. Conidia hyaline, aseptate, smooth-walled, ovoidal or dacryoid to ellipsoidal, upper wedge-shaped, base round, single, straight, 5.2-9.4 × 3.0-4.7 μm (mean = 7.3 ± 0.9 × 3.7 ± 0. 4 μm, n = 30), L/W ratio = 2.0. Culture characteristics -Colonies on MEA reaching 30 mm diam after 14 d at 25 °C in the dark, on OA and PDA reaching 41 mm and 29 mm diam, respectively. OGT 20 °C and MGT 35 °C (Fig. 5). Colonies on MEA white, margin covered with white and velvety aerial mycelium, reverse white. Colonies on OA with a smooth margin, flat, whitish, pale olivaceous in the centre, reverse same colours. Colonies on PDA white, reverse same colours. Notes: Cadophora magna is currently only known from a single isolate (MY902) from soil samples of Mingyong Glacier and is morphologically dis- Page 20 of 29 Zhang et al. IMA Fungus (2022) 13:15 tinct from other Cadophora species in the huge single conidia. In the newly described species, both C. magna and C. inflata produce strongly inflated hyphae cells, but the hyphae cells of C. inflata are often thick-walled and form tuft-like bodies. C. magna is phylogenetically related to Hymenula cerealis, but they are obviously distinguished morphologically, as the latter often produces short chains of spores as well as spores enveloped in a mucus drop (Nisikado et al. 1934). Besides, the placement of H. cerealis should also be confirmed by more molecular data which are currently unavailable.
Colonies on MEA with a weakly undulate margin, brown-grey to yellow-brown, reverse same colours. Colonies on OA with a distinct and white margin, olivaceous to dull green, reverse same colours. Colonies on PDA with a distinct margin, felty, brown, reverse yellow-brown. Notes: Cadophora malorum is a very common Cadophora species and has often been isolated as saprobes or pathogens worldwide (Blanchette et al. 2010;Gramaje et al. 2011;Sugar and Spotts 1992). Strain YL412 was isolated from soil samples collected from Yulong Snow Mountain and the morphological characteristics are similar with the description of the type (Gams, 2000).  Page 24 of 29 Zhang et al. IMA Fungus (2022) 13:15 cal to ellipsoidal conidia that are common in many Cadophora species, but morphologically distincts from the phylogenetically related species of C. indistincta in colony colours and the length of collarettes. Description: Mycelium hyaline, septate, smoothwalled, branched, 1-3 μm wide. Conidiophores hyaline, smooth, often reduced to conidiogenous cells. Conidiogenous cells phialidic, located laterally or terminally, cylindrical or navicular, apex wedge, base truncate, hyaline, smooth-walled, straight or bent, 11.4-25.5 × 1.6-3.1 μm, collarettes evident, 2.1-4.5 μm long, opening 1.6-2.5 μm wide. Conidia hyaline, aseptate, smooth-walled, cylindrical, sporulation abundant, single, straight, 4.5-6.9 × 1.4-2.5 μm (mean = 5.5 ± 0.6 × 1.9 ± 0.3 μm, n = 30), L/W ratio = 2.9. Culture characteristics -Colonies on MEA reaching 36 mm diam, after 14 d at 25 °C in the dark, on OA and PDA reaching 38 mm and 28 mm diam, respectively. Colonies on MEA pale pink to whitish, white at the margin, reverse same colours. Colonies on OA black-grey with light grey margin, reverse same   (Xu et al. 2009) also failed to induce sporulation until we used a slide culture technique. In the multigene phylogenetic tree (Fig. 4), C. yulongensis is closely related to lineages formed by species of Leptodophora and Collembolispora. The genus Leptodophora is currently proposed to accommodate species firstly described as Cadophora. All Leptodophora species produce rarely seceding conidia and the conidial morphology differs markedly (Koukol & Maciá-Vicente, 2022). Species of Collembolispora often produce multicellular macroconidia with appendages and a synasexual morph of phialides (Marvanová et al. 2003). The newly described species is characterized by long cylindrical phialides and cylindrical conidia with comparatively high conidium length/width ratio (2.9).

Discussion
Species of Cadophora have been reported from different locations worldwide, mainly as plant pathogens or root colonizers from northern temperate regions or decomposers from the cold Arctic and Antarctic environments (Blanchette et al. 2004(Blanchette et al. , 2010(Blanchette et al. , 2016(Blanchette et al. , 2021Duran et al. 2019;Gramaje et al. 2011;Maciá-Vicente et al. 2020;Travadon et al. 2015;Walsh et al. 2018). The Qinghai-Tibet Plateau, which is also called "the third pole", is located in the southwest of China and is the highest and largest low-latitude region with permafrost in the world. The unique geographic location of high elevation and low latitude makes the Qinghai-Tibet Plateau a unique alpine ecosystem that is more sensitive to changes of climate and surface conditions (Cheng 1998). Warm, moist air from the Indian Ocean flows up the valleys and is then blocked by huge mountains, leading to abundant rainfall in the southeast range of the plateau. Large numbers of marine glaciers are formed in this area (Shi et al. 1964). During the investigation of cold-adapted fungi from marine glaciers in the Qinghai-Tibet Plateau in 2017, 1208 fungal strains were isolated and identified based on preliminary analyses of generated ITS sequences. Forty-one isolates belonging to Cadophora, one of the three most commonly encountered genera (Cadophora, Geomyces and Pseudogymnoascus; the latter two will be discussed in another paper) were studied in detail. Our results revealed seven Cadophora species, represented by 38 isolates, new to science and three isolates identified to two known species (C. malorum and C. novi-eboraci).
Because of the limited discriminating morphological characteristics existing among Cadophora and related genera, the genus has suffered taxonomic flux since the beginning of its establishment. DNA sequences have provided critical information for species delimitation. Some Cadophora species with multiform morphological characters deviate from the original generic concept, such as C. antarctica, C. fallopiae, C. fascicularis, and C. obovata, have been described mainly based on molecular data (Crous et al. 2017(Crous et al. , 2020Maciá-Vicente et al. 2020). Day et al. (2012) tried to find some consistencies between morphological characteristics and phylogenetic relationships in Cadophora and the related genera. They hypothesized that the ancestral state for these taxa was the production of sclerotium-like heads of multiple phialides and clades derived from phialide arrangements agreed with those generated from rDNA ITS sequence analyses. Although ITS is useful for most fungal species identification, it often fails to discriminate species or even results in misleading information in this group. For example, according to the ITS analyses, Cadophora malorum CBS 165.42 is nested within the Cadophora luteo-olivacea clade, but in the TEF tree, C. malorum CBS 165.42 is strongly supported as the sister group to C. luteo-olivacea (Travadon et al. 2015) and the RPB1 gene can also resolve species relationships between C. meredithiae and C. interclivum better than the ITS (Walsh et al. 2018); C. microspora only known from the sexual morph, was first identified based on ITS and morphological characteristics by Ekanayaka et al. (2019), but in recent studies, it was transferred to Rhexocercosporidium based on LSU and ITS analyses (Hyde et al. 2020). With more genes and species included, Maciá-Vicente et al. (2020) provided a more comprehensive overview about the ecology, morphology and phylogeny of Cadophora. Their results show that the genus is apparently paraphyletic and encompasses a broad spectrum of morphologies and life-styles. They tended to split the genus into three genera: one included those referred to as 'Cadophora s. str. species' that evolved from an ancestor with phialidic conidiogenesis; the second included species like C. interclivum, C. meredithiae, C. luteo-olivacea, C. malorum, and C. helianthi that produced conidia phialidically but are clustered in a separate clade; the third genus should take the name of Collembolispora including Cadophora species with holoblastic conidiogenesis. But this drastic restructuring still needs to be confirmed. Our multi-gene phylogenetic analyses confirmed paraphyly in Cadophora and all the species involved are clustered into two main clades (Fig. 4). Clade 1 comprised 21 Cadophora species (including five newly described in this study and the type species of the genus) and three species belonging to other genera (Hymenula cerealis, Mollisia cinerella, and Phialophora dancoi). This clade was similar to the 'Cadophora s. str. ' clade defined by Maciá-Vicente et al. (2020), just with more species involved in our study. Although all species in Clade 1 have phialidic conidiogenesis, it is somewhat arbitrary to combine P. dancoi, M. cinerella, and H. cerealis into Cadophora at present, as we have just assembled the ITS data sets of these three species to maximize taxon coverage and more exact morphological examinations also need to be done for these fungi. Clade 2 includes most members of Ploettnerulaceae and the remaining Cadophora species. Cadophora constrictospora, C. gregata, C. helianthi, C. interclivum, C. luteo-olivacea, C. malorum, C. meredithiae, C. sabaouae, and C. vivarii which have phialidic conidiogenesis cluster with species including C. antarctica, C. fallopiae, C. inflata, C. obovata, and two species of Mastigosporium which produce conidia with putative enteroblastic or holoblastic conidogenesis. Specimens of C. lacrimiformis only known by the sexual morph is also in this lineage; Leptodophora gamsii, L. echinata, L. orchidicola, L. variabilis, and Collembolispora disimilis which are currently transferred from Cadophora form a subclade with C. yulongensis and two species of Collembolispora; Cadophora fascicularis clusters with species of Mycochaetophora in a distinct lineage. Thus, the currently circumscribed genus could be split into separate genera, but the introduction of more satisfying generic concepts depends on more phylogenetically related taxa in Ploettnerulaceae being involved.
Although Cadophora species are often encountered in cold environments, especially in the polar regions, most of them are psychrotolerant and have an optimum growth temperature (OGT) near or above 20 °C (Blanchette et al. 2021). The only psychrophilic species reported is C. antarctica which was isolated from a soil sample in King George Island (Antarctica) and had an OGT of 15 °C (Crous et al. 2017). Travadon et al. (2015) hypothesized that the geographic distribution patterns of Cadophora species in North America might reflect their adaptation to the contrasting environments: species recovered from cooler areas normally had an OGT at 20 °C and ones isolated from warmer regions tended to grow well at 25 °C. In our study, strains isolated from samples of Dagu Glacier (DG5, DG21, DG1048, DG1073, DG1087, DG1105 and DG1156), Mingyong Glacier (MY527, MY588, MY589, MY873) and Yulong Glacier (YL73) all had optimum growth rates at 20 °C, while others isolated from the same sampling sites had an OGT at 25 °C. Besides, strains being identified as the same species (C. qinghai-tibetana) have different OGTs (ranging from 20 °C to 25 °C). Environmental adaptations of fungal strains might be affected by many factors, such as temperature, humidity, radiation, and substrates. They have to evolve complex abilities to survive in adverse environments. Therefore, it is necessary to test more physiological, biochemical characteristics or perform genome analyses to illustrate adaptation mechanisms of this important fungal group.

Conclusions
Our study shows a very high diversity of Cadophora in the marine glaciers of Qinghai-Tibet Plateau and we described seven Cadophora species new to science. With more species involved, the genus has become apparently paraphyletic and requires phylogenetic reconstruction. Thus, more comprehensive sampling is necessary for the creation of new generic concepts which could accommodate species which deviate morphologically and phylogenetically in this important fungal group.